Sterilization of fluids is an essential step in the manufacture of many pharmaceutical products and foodstuffs. Its goal is the reliable elimination of microorganisms, including viruses, while preserving, as intact as possible, the desirable components of the products. Sterilization may be required of biological fluids, such as nutrient media for fermentation, various blood products, and fluids bearing active pharmaceutical proteins. In the food industry, sterilization of fluid such as milk products is common.
In terms of food sterilization, the selection of a particular sterilization technique frequently is governed by how the procedure will affect the shelf life or the palatability of the food. While the greatest concern in the food industry is bacterial or fungal contamination, dairy products also may carry the additional risk of viral or prion contamination. Elimination or inactivation of such microorganisms is a prerequisite to commercial distribution of these products.
In contrast to the food industry, the choice and use of a sterilization technique in the pharmaceutical industry is subject to the strict demands and regulations imposed upon all pharmaceutical agents that are to be directly administered to an animal or human. There is particular concern about contamination of biological fluids such as pharmaceutical products by viruses, which may be co-isolated from a natural source or introduced during a biotechnological process. For the sterilization of pharmaceutical products, a multi-step process historically has been employed to inactivate, or remove, or reduce viral contaminants. Each step in the process is based on different operational principles to ensure a reduction in the viral load within a fluid preferably by at least four orders of magnitude while preserving the viability of proteins and other desirable components of the fluid.
Irradiation of biological and other fluids with ultraviolet (UV) light has been employed as a method for inactivating undesirable microorganisms. Irradiating plasma and blood products, for example, with UV-light to inactivate viruses was known during WW II. UV-treatment of blood derivatives is especially useful for treating uncoated, heat-stable viruses. Thus, Chin et al., Photochem. & Photobiol. 65, 432-435 (1997) teaches that irradiation of plasma products with UV-light leads to inactivation of the hepatitis A virus and parvoviruses.
UV-irradiation may inactivate microorganisms and/or viruses by generating mutagenic alteration of their genetic material. Above a minimum dose of radiation, the microorganisms lose their reproductive capacity. UV-irradiation damages nucleic acid by creating intrastrand nicks and inducing nucleotide photodimerization, both of which disrupt nucleic acid replication. Through such mechanisms, UV-irradiation can be an effective means of inactivating undesirable microorganisms within biological and other fluids. Unfortunately, the energy of short wavelength UV light also can damage sulfur-containing cysteine bridges and methionine peptide bonds and induce aromatic amino acid side reactions, thereby disrupting the structural and functional integrity of the very proteins that often are the desired end-products of the irradiated fluid. Thus, an inherent problem in the application of UV-irradiation techniques is controlling the irradiation of a fluid so as to ensure sufficient radiation exposure to inactivate undesirable microorganisms within a fluid while at the same time minimizing or eliminating UV-radiation damage to desirable proteins and other components within the fluid.
Traditionally, UV reactors have been used for the UV sterilization of biological fluids. Generally, a UV reactor includes a source of UV radiation such as, for example, one or more elongated tubular bulbs or lamps. In one configuration, an annular reaction chamber with a predetermined width is formed around and encloses the lamp and fluid to be irradiated is pumped or otherwise moved through the chamber, where it is exposed to UV light from the lamp. In another configuration, a UV source or sources may surround and radiate inwardly into a central tubular reaction chamber. In either case, flow rate, light intensity, chamber width or diameter, and reactor length are selected for a particular fluid to ensure, as much as possible, the most effective UV radiation dosage for deactivating undesirable microorganisms while conserving the viability of the desirable components of the fluid.
A problem with the use of UV reactors for irradiating fluid with ultraviolet light results from the finite width of the reaction chamber and the laminar nature of the fluid flow along the chamber. More specifically, as the fluid flows along the chamber, the UV radiation intensity in the treated fluid decreases relatively rapidly as a function of distance from the radiation source. This is due to many factors including the natural inverse-square law of radiation intensity as a function of distance from a source and the absorption characteristics of the fluid and the proteinaceous material supporting the infectious particles. In any event, microorganisms and viruses within layers of the fluid that flow along the outside of the reaction chamber farther from the radiation source receive no or a reduced dosage of radiation. These microorganisms are, therefore, inactivated slowly or not at all. On the other hand, microorganisms in layers of fluid that flow along the inside of the reaction chamber closest to the radiation source receive increased dosages, and in many cases overdoses, of radiation, which, in some cases, is high enough to cause significant damage to desirable proteins and other components in these layers of the fluid. The result is unpredictable and inefficient sterilization and higher levels of damage to desirable components.
Attempts to address these limitations have led to the development of thin-layer or thin film UV reactors in which the width of the reaction chamber and thus the thickness of the fluid layer adjacent the UV source is maintained relatively thin to reduce the detrimental effects of radiation intensity gradients in the fluid (see e.g. Kallenbach et al., Cur. Stud. Hematol. Blood Transfus. Basel 56, 70-82, (1989); Habel et al., J. Immunol. 56, 273-279(1947); Milzer et al., J. Immunol 50, 331-340 (1945). Oppenheimer et al., Am., J. Pub. Health. 49, 903-923, (1959)). The goal is to ensure that all of the fluid is constrained to a region of relatively smaller radiation intensity change as it moves along the radiation source. Thus, the difference in intensity at various layers within the fluid flow is theoretically controlled.
While thin-film reactors have been somewhat successful on a smaller scale, they are problematic in that they can only be scaled up to industrial production throughput with difficulty. This is because keeping the film thickness small and constant can only be realized by increasing the diameter of the reactor and thereby increasing the cross-sectional area of the film to accommodate the desired higher throughput. On an industrial scale, this necessary condition leads to unmanageably large reactors. One attempt to circumvent this problem is suggested in U.S. Pat. No. 5,133,932 which discloses a cylindrical thin-film UV-irradiation reactor in which the area of the film exposed to the UV-light is increased by corrugating the surfaces of the reaction chamber. However, the realized increase in throughput with such a device is marginal at best and still insufficient to accommodate large scale industrial production.
A further limitation of and problem with traditional UV-irradiation reactors is the unfavorable flow profile and dynamic conditions of fluid films when in laminar flow along the radiation source. More specifically, in a laminar flow there is no or very little fluid exchange normal to the flow direction. Thus, as mentioned above, fluid layers farther from the source receive a smaller radiation dose than fluid layers close to the source. Furthermore, the flow velocity profile within a confined laminar flow is such that the flow velocity is relatively low adjacent to the walls of the reaction chamber and is substantially higher intermediate the walls. Thus, fluid closest to the wall of the reaction chamber adjacent the light source flows more slowly and is exposed to the UV radiation substantially longer than fluid between the walls of the reaction chamber. Accordingly, to produce the minimum radiation dose necessary for inactivation of microbial contaminants in the most rapidly flowing fluid layers, the average residence time of the fluid in the reactor must be increased. This leads, however, to increased radiation dosage in the slower moving boundary layers of the fluid flow and consequent increased probability of undesired damage to desirable components in these layers. Thus, destruction of desirable components in the boundary layers due to overexposure is virtually inevitable.
One adverse result of overexposure in some layers of the fluid is the generation of free radicals, which become entrained in the flow and which have adverse effects on desirable components of the fluid. Attempts to minimize damage caused by free-radical generation as a result of overexposure typically include the use of free-radical scavengers in the fluid. Earlier studies have suggested that the use of free-radical scavengers can reduce indirect damage to proteins (Chin et al., Photochem. Photobiol. 65, 432 (1997). Chapman et al. in U.S. Pat. No. 5,922,278 discloses a UV-irradiation sterilization of biological fluids wherein free radicals are scavenged by a scavenging agent. Clark et al. in U.S. Pat. No. 5,786,598 discloses high intensity pulses of short wavelength light to deactivate microorganisms. Morgalis-Nunno et al., U.S. Pat. No. 6,087,141, discloses the use of light in the wavelength range of 340-400 nm (UVA) rather than short wavelengths of about 280 nm or less. Protection of the desired functionality of the fluid is afforded by adding a free-radical scavenger in the form of psoralen. Morowitz et al., U.S. Pat. No. 5,981,163 teaches the addition of quenching protective agents during irradiation deactivation of viruses. While such techniques attempt to deal with the free-radicals generated in the fluid, none address the problems, such as overexposure, that result in the formation of such free-radicals in the first place.
The disruption of the laminar fluid flow through UV reactors has been proposed as a solution to some of the forgoing problems. For example, tangential-flow ring-slot reactors have been proposed as a means to disrupt and induce mixing within the laminar flow layers of a UV reactor. EP 803472 A1 discloses a reactor for UV irradiation of a fluid having an annular or ring-slot reaction chamber surrounding a UV radiation source. The fluid inlet into the reaction chamber is orientated so that the fluid enters tangentially into the chamber in hopes of generating fluid cross-mixing. U.S. Pat. No. 5,433,738 discloses an irradiation reactor for the irradiation of water that includes a helical guide with circular cross section in hopes of generating fluid cross-mixing.
The tangential inflow solution has proven problematic in that the fluid flow through the reaction chamber rapidly reverts, due to wall friction and other hydrodynamic factors, to a fully axial and laminar profile directed along the longitudinal axis of the chamber. The Dean vortices, which are theoretically postulated at least for the area of tangential inflow, and which are intended to promote cross-exchange of the reaction medium within the reaction chamber, are surprisingly not present according to visual studies and CFD-investigations (flow simulation). Tangential entry ring-slot reactors, therefore, afford only a limited solution to the problems discussed above.
A need therefore exists for a method of sterilizing a fluid such as a biological fluid with UV radiation that ensures adequate exposure to inactivate undesirable microorganisms, while simultaneously minimizing or eliminating damage to desirable components in the fluid.
A further need exists for an improved method of inactivating microorganisms in a fluid reaction medium with UV radiation that eliminates the need to use free radical scavenging or quenching agents.
There is also a need for a method of sterilizing biological fluids that is effective at deactivating undesirable microorganisms while preserving the viability of desirable components without the use of scavengers and that is scalable to commercially viable production throughput.
It is to the provision of a method that addresses these and additional needs that the present invention is primarily directed.